Every year, approximately 12,000 people in the United States are burned severely enough to require skin grafting. Diabetic and venous ulcers, as well as pressure sores affect an additional 3 to 4 million people in the United States each year. While engineered tissue analogs have achieved some clinical success as substitutes for damaged skin, prolonged healing times for regenerated skin and mechanically-induced graft failure remain persistent problems. To address the limitations of bioengineered skin substitutes requires an understanding of the mechanisms by which their three-dimensional microarchitecture and biochemical composition mediates keratinocyte adhesion, proliferation and differentiation, and the regeneration of highly functional epithelial tissue. The overall goal of this project is to quantitatively analyze the microstructure and extracellular matrix (ECM) cues that direct keratinocyte functions which promote the rapid regeneration of a robust epidermal layer on the surfaces of precisely engineered basal lamina analogs. We hypothesize that microfabricated basal lamina analogs with three-dimensional features and ECM cues that mimic the cellular microenvironments of the dermal-epidermal junction will increase epidermal stem cell clustering on the surface of bioengineered skin substitutes and promote the rapid regeneration of a robust epidermal layer. To systematically test this hypothesis, we will culture keratinocytes on the surfaces of skin substitutes containing fibroblasts and basal lamina analogs with microfabricated topographic features and discrete ECM compositions, and we will use quantitative morphometric assays to analyze cellular responses to topographic cues and fibroblast paracrine signaling. As such, we propose the following Specific Aims: Specific Aim 1: Establish quantitative relationships between keratinocyte functions and the ECM biochemistry on the surfaces of basal lamina analogs. Specific Aim 2: To determine quantitative roles of scaffold microarchitecture and fibroblast cell signaling on keratinocyte functions as well as on epidermal regeneration on the surfaces of microfabricated basal lamina analogs. The expected outcome is the identification of a series of parameters critical for improving the design of bioengineered skin substitutes, as well as for promoting the rapid regeneration of highly functional skin tissue with increased structural and mechanical stability. To address the limitations of bioengineered skin substitutes requires an understanding of the mechanisms by which their three-dimensional microarchitecture and biochemical composition mediates keratinocyte adhesion, proliferation and differentiation, and promotes the rapid regeneration of robust epithelial tissue. The overall goal of this project is to quantitatively analyze the microstructure and extracellular matrix (ECM) cues that direct keratinocyte functions which promote the rapid regeneration of a robust epidermal layer on the surfaces of microfabricated tissue analogs. Ultimately, we anticipate that the findings from these studies will provide us with parameters for improving the design of bioengineered skin substitutes that will facilitate healing of challenging wounds such as burns and diabetic ulcers. [unreadable] [unreadable] [unreadable]